Exogenous fatty acids affect membrane properties and cold adaptation of Listeria monocytogenes

Listeria monocytogenes is a food-borne pathogen that can grow at very low temperatures close to the freezing point of food and other matrices. Maintaining cytoplasmic membrane fluidity by changing its lipid composition is indispensable for growth at low temperatures. Its dominant adaptation is to shorten the fatty acid chain length and, in some strains, increase in addition the menaquinone content. To date, incorporation of exogenous fatty acid was not reported for Listeria monocytogenes. In this study, the membrane fluidity grown under low-temperature conditions was affected by exogenous fatty acids incorporated into the membrane phospholipids of the bacterium. Listeria monocytogenes incorporated exogenous fatty acids due to their availability irrespective of their melting points. Incorporation was demonstrated by supplementation of the growth medium with polysorbate 60, polysorbate 80, and food lipid extracts, resulting in a corresponding modification of the membrane fatty acid profile. Incorporated exogenous fatty acids had a clear impact on the fitness of the Listeria monocytogenes strains, which was demonstrated by analyses of the membrane fluidity, resistance to freeze-thaw stress, and growth rates. The fatty acid content of the growth medium or the food matrix affects the membrane fluidity and thus proliferation and persistence of Listeria monocytogenes in food under low-temperature conditions.

Because the cells' menaquinone-7 (MK-7) content was previously associated with membrane fluidity under low-temperature conditions, we analyzed this lipid for all cultures grown at 6 °C (Table 1). L. monocytogenes strain DSM 20600 T , FFH, and FFL 1 had an MK-7 content of 213 ± 12, 181 ± 9, and 89 ± 5 nmol g −1 , respectively, after growth in TSB-YE at 6 °C (Table 1). This MK-7 content is in accord with previous findings that the former two strains increase their production under low-temperature growth conditions, but the latter strain does not 26 . After growth in TSB-YE supplemented with P80, strains DSM 20600 T and FFH had significantly lower MK-7 content of 175 ± 16 and 135 ± 5 nmol g −1 , respectively, while strain FFL 1 showed unchanged MK-7 content of 90 ± 8 nmol g −1 . After growth in TSB-YE supplemented with P60 or P60P80, strain FFL 1 was the only strain that significantly increased MK-7 content of 139 ± 2 and 135 ± 8 nmol g −1 , respectively. For the other two strains, supplementation with P60 or P60P80 did not increase the MK-7 content. Thus, supplementation with exogenous fatty acids also affects the fatty acid synthesis of L. monocytogenes. We used the ratio of anteiso-C 15:0 to anteiso-C 17:0 (Ri ai-15/ai-17 ) to assess the impact of exogenous fatty acids on the profile of endogenously synthesized fatty acids. For all supplemented cultures, except one, we noticed the decrease of the Ri ai-15/ai-17 . Strain L. monocytogenes DSM 20600 T grown at 6 °C and supplemented with P60 was the only exception. Thus, the WAMT of the total fatty acid profiles was not primarily determined by the shift of the Ri ai-15/ai-17 but by the T m of the incorporated exogenous fatty acids (Tables 1, 2, 3).
The incorporation of exogenous fatty acids alters membrane fluidity and support cold adaptation. We measured changes in the lateral diffusion capability of the cytoplasmic membranes induced by supplementation with P80 or SSE after growth at 6 °C based on trimethylammonium diphenylhexatriene (TMA-DPH) anisotropy (Fig. 2). The data showed an apparent discrepancy between cells grown with and without exogenous fatty acid supplementation. Supplementation with an exogenous fatty acid source during growth at 6 °C resulted in a higher fluidity of the membranes for all L. monocytogenes strains in a temperature range between 5 and 15 °C. The difference between supplemented and non-supplemented strains is > Δ0.010 at these low temperatures. At higher temperatures, membrane fluidity increased steadily until TMA-DPH anisotropy values for all three strains approximate each other at temperatures of 20 °C and above. L. monocytogenes strains grown at 6 °C in TSB-YE with P80 or SSE as an exogenous source of fatty acids showed a significantly smaller TMA-DPH anisotropy change over the entire measuring range, which indicates a broad transition phase. Strain FFL 1 showed the most considerable effect from all three strains. Growth with P80 and SSE increased cell membrane fluidity and a broader transition phase than strains without supplementation by exogenous fatty acids. This is because these supplements provide fatty acids with low T m . Long-term incubations for up to 48 h of non-growing cells suspended in Ringer's solution with P80 at 6 °C showed no effect on membrane fluidity (Fig. 3). These results indicated the dependency of membrane effects on growing, biochemically active cells but not on the mere abiotic association of P80 with the cell membrane. In addition, for this long-term incubation, C 18:1 could not be detected in the cells' fatty acid profile, indicating that fatty acid profile analyses show only incorporated fatty acids but no fatty acids bound to polysorbate.
Exogenous fatty acids affect resistance to freeze-thaw stress and growth rates. We applied a freeze-thaw stress test as an indicator of membrane integrity. This test showed a positive and a negative impact of exogenous fatty acids sources on cell resistance depending on the supplement (Fig. 4). After growth at 6 °C and subjected to freeze-thaw stress, all L. monocytogenes strains showed a significant decrease of log 10 -reduction of CFU mL −1 if supplemented with P60, P80, or SSE compared to non-supplemented TSB-YE. Strains DSM 20600 T and FFL 1 grown in TSB-YE with P60 and all strains grown in TSB-YE with P80 showed significantly decreased log 10 -reduction after freeze-thaw stress compared to cultures in un-supplemented TSB-YE. Thus, sup- Table 3. Fatty acid (FA) composition, weighted-average melting temperature (WAMT), and the ratio of anteiso-C 15:0 to anteiso-C 17:0 (Ri ai-15/ai-17 ). Tryptic soy broth-yeast extract medium supplemented with food lipid extracts and Listeria monocytogenes strains DSM 20600 T , FFH, and FFL 1 grown at 6 °C on tryptic soy agaryeast extract medium supplemented with 0.1% (wt/vol) milk extract (ME), with 0.1% (wt/vol) minced meat extract (MME) or with 0.1% (wt/vol) smoked salmon extract (SSE). Values are means ± standard deviation (n = 3). 2.0 ± 0.0 5.2 ± 0.9 6.2 ± 0.6 www.nature.com/scientificreports/ plementation with exogenous fatty acids can positively affect cell fitness, regardless of the T m of the incorporated fatty acids. The highest resistance against freeze-thaw stress was observed for strain DSM 20600 T after growth in TSB-YE with P60 and for strains FFH and FFL 1 after growth in TSB-YE with P80 or SSE. Supplementation with SSE produced the exact extent of log 10 -reduction of CFU mL −1 as supplementation with P80 for all strains.  www.nature.com/scientificreports/ The supplementation experiments showed a clear impact on growth rates of the tested L. monocytogenes strains at 6 and 37 °C (Fig. 5). The growth rates at 6 °C were reduced after supplementation with P60 and increased after supplementation with P80, compared to non-supplemented controls. The growth rates decreased from 0.034 to 0.010, 0.048 to 0.013, and 0.048 to 0.017 after supplementation with P60 and increased to 0.047, 0.072, and 0.061 after supplementation with P80 in strains DSM 20600 T , FFH, and FFL 1 grown at 6 °C, respectively. In contrast to cultures grown at 6 °C, an increase of growth rates could be demonstrated for all strains at 37 °C after supplementation with P60 and with P80. Growth rates increased from 0.72 to 0.95 and 0.93 for strain DSM 20600 T , from 0.61 to 0.97 and 0.94 for strain FFH, and from 0.63 to 0.96 and 1.0 for strain FFL 1, grown at 37 °C after supplementation with P60 or P80, respectively. Thus, the exogenous fatty acid with high T m (C 18:0 ) inhibits growth at 6 °C for all strains, whereas a fatty acid with low T m (C 18:1 cis 9) positively affected growth rates. In contrast, both types of fatty acids positively affected growth rates at 37 °C. These growth rates are in accord with our observation that all three strains showed faster colony formation at 6 °C growth temperature on www.nature.com/scientificreports/ tryptic soy agar-yeast extract medium (TSA-YE) if supplemented with ME, MME, or SSE, reflecting the positive influence of exogenous fatty acids from foods.

Discussion
Some foods are known to have an increased risk for contamination with L. monocytogenes even under lowtemperature storage conditions 1,12 . We used P60 with a high T m fatty acid (C 18:0 , T m 69.2 °C) and P80 with a low T m fatty acid (C 18:1 cis 9, T m 12.8 °C) as supplements. Straight chain fatty acids, saturated and unsaturated, were not synthesized by L. monocytogenes. Therefore the incorporated C 18:0 and C 18:1 cis 9 must be exogenous in origin [27][28][29][30] . The same is true for other unsaturated fatty acids supplemented with the food extracts such as C 18 www.nature.com/scientificreports/ growth temperatures than strain FFL 1 (Tables 1, 2). As described before, MK-7 is an additional modulator of membrane fluidity for these strains and is crucial for bacterial cell fitness 26,31 . However, all strains showed an expansion of their fatty acid profiles after supplementation with exogenous fatty acids as these were assimilated by all L. monocytogenes strains (Tables 1, 2, 3). All strains were even able to incorporate polyunsaturated fatty acids such as C 20:5 with a T m of − 53 °C and C 22:6 with a T m of − 44 °C derived from the supplemented SSE. The bactericidal effects of polyunsaturated fatty acids 32 , as previously described, did not occur in L. monocytogenes. As expected, exogenous fatty acids with lower T m and those with higher T m such as C 14:0 with a T m of 53.5 °C and C 16:0 with a T m of 62.2 °C were incorporated. We found no indication for selective incorporation of particular fatty acids. The supplementation with an equimolar mixture of P60 and P80 showed no favored incorporation of the lower melting point fatty acid in all strains at low-temperature growth conditions. All strains did not selectively incorporate the supplemented fatty acids according to their T m , but their percentage availability in the medium (Tables 1, 2, 3). This finding is also in accord with the increasing appearance of exogenous fatty acids in the fatty acid profiles of the strains during cultivation (data not shown). Exogenous fatty acids replaced endogenously synthesized fatty acids and affected the fatty acid synthesis of L. monocytogenes. We found a decrease of the Ri ai-15/ai-17 in the presence of exogenous fatty acids, which indicated the stimulation of chain elongation during the synthesis of branched-chain fatty acids. The reduction of the Ri ai-15/ai-17 was related to the presence of exogenous fatty acids but not to the nature of these fatty acids. Although the shift to longer branched-chain fatty acids should increase the membrane melting temperature, we found WAMT values primarily affected by the melting temperatures of exogenously supplied fatty acids. WAMT values increased in all strains after supplementation with P60 due to the presence of C 16:0 and C 18:0 (Table 1). In addition, we detected significant differences for WAMT and MK-7 content between tested strains supplemented with P60 and P80. For L. monocytogenes strains DSM 20600 T and FFH, a reduced MK-7 content was detected when C 18:1 cis 9 was incorporated and WAMT decreased. In contrast, strain FFL 1, which was previously reported not to have an MK-mediated temperature adaptation, increased MK-7 content in the presence of C 16:0 and C 18:0 . These results support the previous evidence that fatty acids are not selectively incorporated as WAMT increased after supplementation with P60 compared to the non-supplemented cultures. Furthermore, our data demonstrate that the previously described MK-mediated adaptation of membrane fluidity 26 and cell fitness 31 in L. monocytogenes is affected by low growth temperatures and the presence of exogenous fatty acids. Thus, the interlocking of MKmediated adaptation and FA-dependent cold adaptation is more complex as expected, as exogenous fatty acids can impact the MK content and, therefore, are also involved in the cold adaptation of L. monocytogenes.
A critical feature of this study was to confirm the incorporation of exogenous fatty acids into membrane lipids. L. monocytogenes cannot synthesize unsaturated fatty acids [27][28][29][30] . Therefore, C 18:1 covalently linked to phospholipids PG and LPG as revealed by total Q-TOF MS must be exogenous in origin (Fig. 1a,b). All phospholipids analyzed contained one C 15:0 acyl chain combined with a second acyl chain (C 15:0 , C 18:1 , C 16:0, or C 17:0 ). PG and LPG species containing C 15:0 /C 15:0 or C 15:0 /C 18:1 were the dominating molecular species (Fig. 1c-f). Two different acyltransferases are involved in the synthesis of phospholipids in bacteria. A glycerolphosphate acyltransferase synthesizes lysophosphatidic acid, and a lysophosphatidic acid acyltransferase produces phosphatidic acid, the precursor of all phospholipids 17 . Due to the presence of C 15:0 in all phospholipids of L. monocytogenes, one of the two acyltransferases may be characterized by a high substrate specificity for C 15:0 . In contrast, the other acyltransferase may show a broader substrate specificity for different fatty acids. Furthermore, a high similarity between the molecular species distribution regarding PG and LPG could be observed. Besides, we could detect neither C 18:1 nor C 18:0 in glycolipids, which are present in L. monocytogenes in addition to phospholipids 33 . Therefore, all these observations favor the enzymatic and probably selective (concerning the sn-position of the glycerol) incorporation of high amounts of C 18:1 and conclusively of other exogenous fatty acids into phospholipids. These results suggest that other detected exogenous fatty acids of the fatty acid profile are also covalently bound to the polar lipids of L. monocytogenes, contrasting a previous report, which found no integration of C 18:1 into phospholipids www.nature.com/scientificreports/ only the intercalation of this lipid in the bacterial membrane 34 . This discrepancy may be attributed to the use of ester-bound fatty acids (P60, P80, food extracts) in our study, in contrast to free fatty acids as a supplement in previous studies. We did not study the polar lipids in more detail, as the polar head groups of the membrane lipids have only minor effects on the thermal membrane properties and show no changes in their composition in L. monocytogenes at low growth temperatures [35][36][37] . Fatty acids with a high T m (saturated, straight-chain fatty acids) decrease membrane fluidity, whereas fatty acids with a low T m (unsaturated and branched-chain fatty acids) increase membrane fluidity 38 . TMA-DPHdependent anisotropy measurements confirmed the influence of exogenous fatty acids on the membrane fluidity of whole living cells with a complex lipid composition (Fig. 2). None of the strains showed the two typical plateaus that indicate the two temperature-dependent ultimate states of biomembranes: the gel-like solid-state (high TMA-DPH anisotropy) and the liquid-crystalline liquid-state (low TMA-DPH anisotropy). A linear curve progression rather than a sigmoidal curve described the relation between anisotropy and measuring temperature for all strains tested, which generally describes the phase transition of the membrane 39 . The cultures grown in TSB-YE supplemented with P80 or SSE showed a higher membrane fluidity in all strains than those without supplementation. These results indicated significantly more fluid membrane below 20 °C and unchanged fluidity www.nature.com/scientificreports/ above 20 °C. The change in TMA DPH anisotropy with a value of 0.03 was approximately the same for all three strains. After supplementation with P80 and SSE (Tables 1, 3), the altered fatty acid profile suggests that the exogenous unsaturated fatty acids with low T m cause this effect, resulting in more beneficial membrane fluidity and more pronounced adaptation of the membrane to low temperatures. Because washed cells incubated in Ringer's solution and supplemented with P80 and SSE did neither show the implementation of supplemented fatty acids nor any change in TMA DPH anisotropy, we concluded that active incorporation of exogenous fatty acids in growing cells is a prerequisite for impacting cell membrane fluidity by these exogenous lipids (Fig. 3).
We also demonstrated that cell membranes complemented with exogenous and low T m fatty acids, such as C 18:1 cis 9, are protective against freeze-thaw stress. Resistance against freeze-thaw stress is used to indicate a resilient and robust membrane structure 40 . Significantly higher resistance was detected as log 10 -reduction of CFU mL −1 for all strains grown at 6 °C with incorporated C 18:1 cis 9. In contrast, only strain DSM 20600 T showed lower log 10 -reduction of CFU mL −1 after C 16:0 and C 18:0 incorporation. The strains FFH and FFL 1 showed no significant changes in the log 10 -reduction of CFU mL −1 when C 16:0 was incorporated. Furthermore, bacterial cell growth was reduced after incorporation of C 16:0 and C 18:0 and increased after the incorporation of C 18:1 in all strains grown at 6 °C. On the other hand, if cells were grown at 37 °C, both fatty acids increased the growth rate for all strains (Fig. 5).
Yao et al. 17 stated that L. monocytogenes do not actively incorporate exogenous fatty acids into their membrane phospholipids. Nevertheless, the genome encodes the gene loci lmo1814, lmo1863, and lmo2514, representing homologs of the two-component fatty acid kinase system FakA/FakB of S. aureus. For S. aureus, this system catalyzes the first steps in exogenous fatty acid incorporation, which is the binding and phosphorylation of exogenous fatty acids. The acyl-phosphates formed can then enter the phospholipid synthesis 20,41 . A standard nucleotide Basic Local Alignment Search Tool (BLAST) check revealed that homologs of the FakA/FakB genes are present in at least 100 deposited genomes of L. monocytogenes, highlighting the conservation of these genes and the crucial importance of this mechanism. Furthermore, we confirmed the presence of lmo1814, lmo1863, and lmo2514 in all strains used in this study by specific PCR and subsequent sequencing analysis of the PCR products.
In this study, we could demonstrate that the fatty acid profile of L. monocytogenes was modified by exogenous fatty acids at low and high growth temperatures, changing membrane fluidity and growth properties. The present exogenous fatty acid improves membrane fluidity and cell viability at low growth temperatures. The influence of external fatty acids from the food matrix significantly affects the contamination dynamics of chilled foods. We demonstrated that the acyl chain composition plays a crucial role in the survival of L. monocytogenes, and an increase in straight-chain fatty acids reduces the organism's growth rate.

Materials and methods
Materials. All chemical reagents and solvents were purchased from Alfa Aesar, Carl Roth, MilliporeSigma, Sigma-Aldrich, Thermo Fisher Scientific, or VWR. All solvents and water for analytics were HPLC grade and used as received. Ultra-high temperature processed milk (3.5% fat), modified atmosphere packaged minced meat, and pre-cut vacuum-packed smoked salmon were purchased at a local supermarket chain store.
Strains, culture media, and cultivation. In this research, three different strains of L. monocytogenes were examined. Strain FFH (= DSM 112142; serovar group 4b, lineage I) was isolated from minced meat in 2011 and strain FFL 1 (= DSM 112143; serovar group 1/2a or 3a, lineage II) from smoked salmon in 2012. In addition, the strain L. monocytogenes DSM 20600 T (serovar group 1/2a, lineage II) was obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. The adaptive response of strain FFL 1 to low temperature is primarily an FA-dependent mechanism, while strains DSM 20600 T and FFH also expressed an MK-based response 26 .
All strains were aerobically cultured in 200 µL or 100 mL TSB-YE. The medium is composed of tryptic soy broth containing 17 g peptone from casein L −1 , 3 g peptone from soy meal L −1 , 2.5 g d-glucose L −1 , 5 g sodium chloride L −1 , and 2.5 g dipotassium hydrogen phosphate L −1 supplemented with 6 g yeast extract L −1 or in BHI broth composed of 12.5 g brain infusion solids L −1 , 5 g beef heart infusion solids L −1 , 10 g protease peptone L −1 , 2 g glucose L −1 , 5 g sodium chloride L −1 and 2.5 g disodium phosphate L −1 using 96-well microplates or 300 mL Erlenmeyer flasks, respectively. The TSB-YE was supplemented with 0.1% (wt/vol) polysorbate 60 (P60), with 0.1% (wt/vol) polysorbate 80 (P80), with each of 0.05% (wt/vol) P60 and P80 (P60P80), with 0.1% (wt/vol) ME, with 0.1% (wt/vol) MME, or with 0.1% (wt/vol) SSE, respectively. 0.078% (wt/vol) d-sorbitol was used as control. We measured the medium's a w with a LabMaster-aw instrument (Novasina, Switzerland). OD 625 documented growth in TSB-YE with or without supplementation by a GENESYS 30 visible spectrophotometer (Thermo Fisher Scientific, USA) or a Synergy H1 modular multimode microplate reader (BioTek Instruments, Inc., USA). Growth was fitted by the Gompertz growth model as previously described 42 . Cultures were prepared in multiple independent replicates, inoculated with 1% (vol/vol) of an overnight culture at 30 °C and incubated on an orbital shaker at 6 or 37 °C and 150 rpm until late exponential phase (OD 625 = 0.8-1.0) or stationary phase for growth rate determination. Cultures were harvested by centrifugation (10 min at 10,000 × g) at growth temperature and washed thrice with sterile phosphate-buffered saline (PBS) pH 7.4. Subsequently, bacterial cells were used for temperature stress tests, fatty acid analysis, determination of MK content, polar lipid analysis, and membrane fluidity analysis. Colonies were cultivated on TSA-YE at 30 °C. Additionally, each strain was incubated on TSA-YE supplemented with 0.1% (wt/vol) ME, with 0.1% (wt/vol) MME, or with 0.1% (wt/vol) SSE for fatty acid analysis.
To determine colony forming units (CFU) for the freeze-thaw stress test, 50 µL of serial dilutions were plated on TSA-YE (90 mm Petri dish) using the exponential mode (ISO 4833-2, ISO 7218, and AOAC 977.27) of the easySpiral automatic plater (Interscience, France). After a one-day incubation at 37 °C, the CFU were counted for the corresponding dilution steps, and the weighted average of enumerated L. monocytogenes was given in CFU www.nature.com/scientificreports/ mL −1 . The results for the temperature stress test were presented as decadic logarithm (log 10 ) reduction relative to the initial CFU mL −1 , respectively.
Lipid extraction from food. Total lipids from commercially available milk (3.5% fat), minced meat, and smoked salmon were extracted using the method by Bligh and Dyer as previously described 43 . Minced meat and smoked salmon were used directly without any pretreatments. Milk was freeze-dried before extraction. One hundred grams of food were incubated for 2 h at room temperature under shaking with 150 mL of chloroform/ methanol (1:2, vol/vol) in a 500 mL Erlenmeyer flask. Then we added 50 mL of chloroform and let the mixture shake for another 1 h. The extract was filtered using cellulose filter paper. For phase separation, 90 mL of PBS (pH 7.4) were added, mixed vigorously, and incubated at − 20 °C. The lower layer was evaporated to dryness with nitrogen and stored at − 20 °C.
Freeze-thaw stress tests. The freeze-thaw stress test was performed by subjecting each strain to three freeze-thaw cycles. First, three aliquots of 2 mL bacterial cell suspension for each strain were frozen at − 20 °C. Then, after 24, 48, and 72 h, aliquots were thawed for 20 min at room temperature, and the number of CFU mL −1 was determined. Finally, the remaining aliquots were refrozen for subsequent freeze-thaw cycles.
Fatty acid extraction and analysis. Approximately 40 mg of washed bacterial cells per sample were used for fatty acid analysis. Fatty acids were extracted and analyzed as methyl esters (FAMEs) as previously described 26 . First, cells were resuspended in 1 mL of 15% (wt/vol) sodium hydroxide (NaOH) in methanol/water (1:1, vol/vol) using 10 mL hydrolysis tubes and saponified for 30 min at 100 °C. Next, fatty acids were methylated with 2 mL (6 N) hydrochloric acid/methanol (1:1.2, vol/vol) for 10 min at 80 °C and immediately cooled on ice. Next, fatty acid methyl esters were extracted with 1.25 mL hexane/methyl tert-butyl ether (1:1, vol/vol) for 10 min in an overhead mixer. Phases were separated by centrifugation (5 min at 3000 × g), and the lower phase was discarded. Subsequently, a base wash of the upper phase was performed with 3 mL of 1.2% (wt/vol) NaOH in water. The fatty acid methyl esters were identified by gas chromatography-mass spectrometry (GC--MS) with a 7890A gas chromatograph (Agilent Technologies, USA) equipped with a 5% phenylmethyl silicone capillary column coupled with a 5975C mass spectrometer (Agilent Technologies, USA), as previously described 44 . Fatty acid analysis was performed with MSD ChemStation software (version E.02.00.493, Agilent Technologies, USA), and their retention times and mass spectra were identified. In addition, dimethyl disulfide (DMDS) derivatization and analyses of unsaturated FAMEs were performed as described by Nichols et al. 45 .
The effect of alterations in fatty acid profiles associated with the supplemented lipids on membrane fluidity was determined by calculating the weighted average melting temperature (WAMT) as described previously 26 . Considering the individual melting temperatures of each FA, this parameter allows integrating the quantitative changes of all membrane-associated fatty acids. However, the WAMT value does not represent the actual melting temperature of the cytoplasmic membrane, which also depends on the total polar lipid structure. Therefore, the melting temperatures for fatty acids were taken from previously described research 46,47 .
The bacterial membrane's weighted average melting temperature (WAMT) was calculated according to equation 1. All fatty acids (FA 1 to FA n ) that are present in the fatty acid profile, FA 1 (%) is the percentage of fatty acid no. 1 and melting temperature (T m ) of the corresponding fatty acid. The difference in WAMT (ΔWAMT) indicates the extent of adaptation through the fatty acid shift. Polar lipid extraction and analysis. The mass spectra of polar lipids were analyzed to verify whether the fatty acids from the supplemented culture media were covalently linked to polar lipids of the bacterial membrane. Total lipids from bacterial cells were extracted according to Bligh and Dyer 48 . Approximately 50 mg bacterial cells were resuspended in 3 mL H 2 O and boiled for 10 min using 10 mL hydrolysis tubes. Ruptured cells were centrifuged (15 min at 3000 × g), and the supernatant was discarded. The extraction was performed in two steps using 3 mL chloroform/methanol (1:2, vol/vol) and 3 mL chloroform/methanol (2:1 vol/vol) under shaking for 30 min. Extracts were pooled, and phases were separated by adding 3 mL chloroform and 0.75 mL water (with a final ratio of chloroform/methanol/water of 2:1:0.75, vol/vol/vol) followed by centrifugation (15 min at 3000 × g). The organic phase was evaporated to dryness with nitrogen and stored at − 80 °C until analysis. For analysis, the evaporated extracts were dissolved in 0.1 mL with chloroform/methanol (2:1 vol/vol) and filtered through 0.2 μm polytetrafluoroethylene (PTFE) filters (VWR International, Germany).
Membrane lipids were analyzed using a 6530 Q-TOF MS (Agilent Technologies, United States) by direct infusion of total lipid extracts in the positive ion mode 49 . PG and LPG were additionally measured in the negative ion mode with 50 V collision energy. Lipids were selected in a non-targeted approach in the "auto-MS/MS" mode, which means that the most intense precursor ions are selected automatically. Glycolipids were separated by solid-phase extraction of total lipid extracts with Isolute SI Columns (Biotage AB, Sweden) before analyzing with Q-TOF MS. The data acquisition was performed with MassHunter software (version B.02.00; Agilent Technologies, USA).
Isoprenoid quinone extraction and analysis. About 30-50 mg cells were extracted with methanol/ chloroform (9:5, vol/vol) as previously described 26,50 . Evaporated extracts were made up to 1 mL with methanol and analyzed using a 1260 Infinity Quaternary LC system (Agilent Technologies, USA) equipped with a quaternary pump, an autosampler, a thermo-controlled column compartment, and a diode array detector. Compounds www.nature.com/scientificreports/ were separated isocratically at 30 °C on a Hypersil™ ODS C18 column (Thermo Fisher Scientific, USA) using methanol/diisopropyl ether (9:2, vol/vol) as eluent (flow rate of 1 mL min −1 ). Isoprenoid quinones were detected at 270 and 275 nm and were identified by their absorption spectrum and retention time. The quinones were quantified as vitamin K 1 equivalents using an external calibration curve and an internal vitamin K 1 standard. Data acquisition was performed with OpenLAB CDS ChemStation software (version C.01.07, Agilent Technologies, USA).

Membrane fluidity analyses by anisotropy.
Whole bacterial cells were prepared and stained with the fluorescent probe TMA-DPH to determine anisotropy, as Seel et al. 26 described. TMA-DPH anisotropy is particularly suitable for measuring membrane fluidity measuring the direct mobility of the probe and adjacent lipids 39 . High TMA-DPH anisotropy values correspond to low fluidity and low values to high membrane fluidity. Steady-state fluorescence was measured in an LS 55 fluorescence spectrometer combined with a PTP-1 Peltier system (PerkinElmer, United States) for sample temperature regulation. Cells were washed and resuspended in Tris-EDTA (TE) buffer solution (pH 7.4) and diluted to OD 625 0.2. TMA-DPH stock solution was prepared in dimethyl sulfoxide (DMSO) at a concentration of 0.4 mM. Cells were stained with 0.5 µM TMA-DPH for 10 min at 30 °C in the dark and washed twice. Measurements were performed with a 2 mL sample volume in 3.5 mL quartz glass cuvettes (Hellma, Germany). For TMA-DPH anisotropy measurements, samples were excited at 355 nm, and emission intensities were recorded at 425 nm. Anisotropy (r) values were calculated from polarized intensities using Eq. (2).